NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. This project investigates how chemical toxins or physical factors alter metabolic processes. A recent objective of this project has been the development of a better understanding of the molecular basis for the physiological and toxicological properties of boron. As shown in earlier studies, serine proteases and mechanistically related enzymes such as gamma-glutamyl transpeptidase interact with borate and alcohols to form ternary complexes that inhibit my mimicry of the transition state. We had previously obtained data indicating that in addition to the active site complex, borate was apparently binding to an additional site on the surface of the trypsin molecule. During the past year, we determined the structure of a covalent complex of borate with the enzyme trypsin, that involves formation of a bidentate covalent complex with surface residues Ser164 and Ser167. Analysis of this interaction leads to predictions of structural characteristics of other proteins that would be consistent with formation of similar borate complexes. In addition to providing insight into the physiological effects of borate, such interactions can in principle form the basis for the development of boronate ligands. [unreadable] [unreadable] A second area of effort has involved the development of improved methods for the measurement of intracellular cations. Since the introduction of 5FBAPTA as an NMR indicator for intracellular calcium, a goal of this research has been the development of a trifluoromethyl analog, which can provide greater sensitivity. During the past year, we synthesized a trifluoromethylated calcium chelator for the measurement of organelle calcium, and are currently evaluating its utility.